36 H. O. HALVORSON 



germinated. In order to throw light upon this problem, we 

 centrifuged the suspension at the conclusion of the germination 

 and added to the supernatant some fresh spores, but none of 

 these germinated. This clearly demonstrated that something had 

 happened to the menstruum. Since there was no change in the 

 total amount of alanine present, we examined the L-alanine to 

 see if some had been converted to D-alanine. This appeared 

 reasonable since Hills^-^ had previously demonstrated a D-alanine 

 interference with germination brought about by L-alanine. A 

 racemic mixture of l and d was found after less than one hour's 

 contact with the spores. This prompted us to look for the 

 enzyme racemase which, fortunately, was very easily found'*. 

 It proved to be an interesting enzyme because it was active in 

 the intact spore and was heat resistant, withstanding tempera- 

 tures up to 100°. Upon more careful study, we found the enzyme 

 was not only heat resistant but remained so even after the spores 

 germinated. Furthermore, this enzyme was attached to some 

 colloidal particles. Upon separation from the carrier by means 

 of a sonic oscillation, it becomes heat sensitive. 



These observations stimulated us and others to look for more 

 enzymes in spores. Previous to this time, most bacteriologists 

 had assumed that spores were devoid of enzymes because most 

 studies made in the past had resulted in negative findings. A 

 variety of enzymes similar to the racemase have now been found 

 in the spores of aerobic bacilli^-^' ^^. A heat-resistant catalase is 

 present in most aerobic spores and also a heat-resistant ribo- 

 sidase, an enzyme which hydrolyzes adenosine into adenine and 

 ribose. The latter enzyme has also been shown to be heat 

 resistant and to be associated with a colloidal particle. The heat- 

 resistant catalase does not appear to be particulate. There is 

 strong evidence to suspect its existence in spores of other heat- 

 resistant enzymes, particularly proteolytic enzymes^'^. These 

 may, in fact, be the ones responsible for the changes occurring 

 during storage and also during heat shock ; for if these are like 

 racemase, they will not be destroyed by the temperatures used 

 for heat sensitization, and the reactions they bring about 



